Immunosenescence caused by age-associated involution of the thymus poses a significant risk to the aging human population. During age-associated thymic involution, a reduction and disorganization of the functional regions of the thymus, along with an increase in adipogenic and other undesirous cell types, results in a lower capacity of the thymus to generate functional T-cells required for adaptive immunity. Currently, the cellular origins of thymic adipocytes remain obscure, with some investigators suggesting that adipocytes infiltrate the thymus during involution and others indicating that they differentiate directly from thymic stromal cells. Our lab has identified a potential regulator of adipogenesis in the thymus, the Activating Transcription Factor 3 (ATF3), in which deletion of the Atf3 gene results in an increased presence of lipid-laden thymic stromal cells at 2- months of age in mice when assessed by both flow cytometry and immunohistochemistry. At 10-months of age, FACs analysis of adipogenic cells in Atf3 mutants and wild-type controls show that they are similar quantitatively. However, qualitative analysis by immunohistochemistry reveals differences in adipogenic cell types in Atf3 homozygous and heterozygous mutants, which warrants further investigation. We have also conducted a lineage trace study using Atf3null/null; Foxn1Cre/+; Rosa26Tom/+ mice to identify if a subset of thymic epithelial cells (TECs) undergo adipogenesis by using imaging flow cytometry. We identified four major classes of TEC-derived adipogenic cells (LipidTox+EpCAM+Ly51+, LipidTox+EpCAM+Ly51-, LipidTox+EpCAM- Ly51-, and LipidTox+EpCAM-Ly51+ cells), which include lipid-laden cTECs and mTECs. In frozen thymic sections, we have identified lipid-laden cTECs that express PPARy starting at 6, 7, and 10-months of age. In addition, we identified adipogenic mTECs that express FSP1, a marker for EMT, when we looked at 10-month old mice. These findings suggest that although both cTECs and mTECs become adipogenic, each may be regulated by different molecular mechanisms. We have also looked at tissue from a PPAR?-tdTomato reporter mouse engineered by the laboratory of Diane Mathis at Harvard. Using the reporter mice, we have identified adipogenic vascular-associated cells and the presence of globular fat cells within the lumen of the vasculature that appear to be infiltrating the thymus. Overall, we have begun to characterize the different classes of thymic adipocytes more fully, and we are beginning to understand more about the genes that may be governing the process. We have optimized techniques to study thymic adipocytes using standard and imaging flow cytometry and immunocytochemistry. We have access to a novel mouse line, the PPARy-tdTomato line, that we plan on using for our research purposes. These data suggest that both cTECs and mTECs give rise to a subpopulation of thymic adipocytes and that ATF3 is a likely repressor of adipogenesis during thymic involution. In Aim 1, we will identify the cell type in which ATF3 acts to regulate thymic adipogenesis. In Aim 2, we will identify the mechanisms by which ATF3 regulates adipogenesis.